Transcriptional Regulators of Exocrine Pancreatic Develo

描述（申请人提供）： 这项研究的目的是确定胰腺转录 建立和维持特定于发展计划的因素 胰腺的外分泌组织区室。我们将开始（具体目标1）， 鉴定介导腺泡细胞的DNA结合转录因子 PTF 1-p48基因转录增强子的活性，一个关键的 腺泡特异性基因的转录激活因子。为此，我们将定义 最小功能增强子，然后通过以下方式绘制核蛋白结合位点： 体外DNA酶I足迹法和基于计算机的进化搜索 特异性转录因子的保守结合序列 家庭我们将确认足迹和共识约束力的相关性 通过分析突变位点对细胞活性的影响， 增强剂我们将确定候选的DNA结合转录因子， 胰腺外分泌7个主要转录因子的筛选 在发育和成熟的外分泌胰腺中表达的家族， RT-PCR方案（具体目标2）。我们会将每个胰腺癌患者 具有潜在p48增强子结合位点的因子 它们所属家族的结合序列。我们会核实 通过测试核苷酸序列是否 绑定的要求与 增强子以及该因子是否可以通过增强子激活报告基因。 转染细胞中的元件。我们将通过审查 它们的表达是否发生在胚胎阶段， 发展的调节作用。最后，我们将测试候选人的角色 显性负性和组成性活性的强制表达 胚胎胰腺雏形中的嵌合形式的因子能够 培养中的形态发生（具体目标3）。

DESCRIPTION (Provided by Applicant): The goal of the proposed research is to identify pancreatic transcription factors that establish and maintain the developmental program specific for the exocrine tissue compartment of the pancreas. We will begin (Specific Aim 1) by identifying the DNA-binding transcription factors that mediate the acinar cell activity of the transcriptional enhancer of the gene for PTFl-p48, a critical transcriptional activator of acinar specific genes. To do this we will define the minimal functional enhancer and then map nuclear protein binding sites by DNase I footprinting in vitro and computer-based searches for evolutionarily conserved binding sequences characteristic of specific transcription factor families. We will confirm the relevance of the footprints and consensus binding sites by analyzing the effects of mutating the sites on the activity of the enhancer. We will identify candidate DNA-binding transcription factors of the exocrine pancreas by screening all members of seven major transcription factor families for expression in the developing and the mature exocrine pancreas by an RT-PCR protocol (Specific Aim 2). We will match individual pancreatic factors with potential p48 enhancer binding sites through the known consensus binding sequences of the family to which they belong. We will verify the match between factor and the enhancer site by testing whether the nucleotide sequence requirements for binding match the sequence requirements for activity in the enhancer and whether the factor can activate a reporter gene through the element in transfected cells. We will screen additional candidates by examining whether their expression occurs at an embryonic stage consistent with a developmental regulatory role. Finally, we will test the role of candidate factors by forced expression of dominant-negative and constitutively active chimeric forms of the factors in embryonic pancreatic rudiments capable of morphogenesis in culture (Specific Aim 3).